Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA–Protein Complexes Using Mismatch Repair Protein MutS as a Model

Molecules

Mayya V. Monakhova¹  Viktor A. Anashkin¹  Elena A. Romanova¹  Elena A. Kubareva¹  Tatiana S. Oretskaya¹  Egor M. Kosaretskiy²  Kirill K. Kolesnikov³  Maria I. Zvereva³  Timofei S. Zatsepin³  Peter Friedhoff⁴ 

[1] A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskye Gory 1, 119991 Moscow, Russia;Department of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Leninskye Gory 1, 119991 Moscow, Russia;Department of Chemistry, Lomonosov Moscow State University, Leninskye Gory 1, 119991 Moscow, Russia;Institute for Biochemistry, FB 08, Justus Liebig University, Heinrich-Buff-Ring 17, D-35392 Giessen, Germany;

关键词: regioselectivity;    DNA modification;    DNA–protein complex;    modified oligonucleotide;    crosslinking;    DNA mismatch repair;   

DOI  :  10.3390/molecules27082438

来源: DOAJ

【 摘 要 】

Covalent protein capture (cross-linking) by reactive DNA derivatives makes it possible to investigate structural features by fixing complexes at different stages of DNA–protein recognition. The most common cross-linking methods are based on reactive groups that interact with native or engineered cysteine residues. Nonetheless, high reactivity of most of such groups leads to preferential fixation of early-stage complexes or even non-selective cross-linking. We synthesised a set of DNA reagents carrying an acrylamide group attached to the C5 atom of a 2′-deoxyuridine moiety via various linkers and studied cross-linking with MutS as a model protein. MutS scans DNA for mismatches and damaged nucleobases and can form multiple non-specific complexes with DNA that may cause non-selective cross-linking. By varying the length of the linker between DNA and the acrylamide group and by changing the distance between the reactive nucleotide and a mismatch in the duplex, we showed that cross-linking occurs only if the distance between the acrylamide group and cysteine is optimal within the DNA–protein complex. Thus, acrylamide-modified DNA duplexes are excellent tools for studying DNA–protein interactions because of high selectivity of cysteine trapping.

【 授权许可】

Unknown